ARTICLES Immunohistochemical Classification and Functional Morphology of Human Choroidal Ganglion Cells Christian Albrecht May,1 Winfried Neuhuber,2 and Elke Lu¨tjen-Drecoll1 PURPOSE. To characterize human choroidal ganglion cells Approximately, 2000 of these neurons are located in the inner (CGCs) further, regarding their immunohistochemical and ul- portion of the ciliary muscle and presumably are involved in trastructural appearance and their pre- and postsynaptic con- fine regulation of the accommodation.3 In addition, approxi- nections. mately 2000 neurons are present in the choroid. Most of the METHODS. Choroidal wholemounts and serial sections of human postganglionic nerve fibers of these choroidal ganglion cells (CGCs) join the perivascular nerve fiber plexus that supports donor eyes were stained with antibodies against neuronal ni- 1,2 tric oxide synthase (nNOS), vasoactive intestinal peptide (VIP), the vasodilative innervation of the choroidal vasculature. A tyrosine hydroxylase (TH), vesicular monoaminergic trans- small number of these CGCs is located within the ciliary porter (VMAT)-2, vesicular acetylcholine transporter (VAChT), nerves. The postganglionic nerve fibers of this group of neu- rons do not enter the choroid but join the nerve fiber plexus neuropeptide Y (NPY), substance P (SP), calcitonin gene-re- 4 lated peptide (CGRP), calretinin, galanin, synaptophysin, and that innervates the outflow system. ␣-smooth muscle actin. Ultrathin sections of glutaraldehyde- When different mammalian eyes were compared, substan- fixed eyes were studied with an electron microscope. tial numbers of CGCs in the posterior choroid were found only in higher primates and human eyes containing a fovea centra- RESULTS. All CGCs stained for nNOS, most for VIP, approxi- lis.5 CGCs are also present in a number of birds with a special- mately 45% for calretinin, and only single neurons for NPY and ized accommodative system and one or more foveae.6,7 Further galanin. Ultrastructurally, the CGCs showed an incomplete characterization of the CGCs in the duck revealed that in glial sheath and, in places, showed close contact to surround- addition to NO and VIP, the CGCs express galanin. The post- ing collagen fibrils. The CGCs were in close contact with ganglionic fibers of these cells innervate not only the choroidal numerous boutons staining for the different neuronal markers vasculature, but also the numerous non–vascular smooth mus- including synaptophysin, nNOS, VIP, NPY, TH, VMAT-2, cle cells (NVSMCs) present in the avian choroidal stroma.8 VAChT, calretinin, and NPY. Duck CGCs are surrounded by tyrosine hydroxylase (TH)/ CONCLUSIONS. The data indicate a complex integrative function dopamine-␤-hydrolase (DBH)–immunoreactive nerve fibers of CGCs. The immunohistochemical and ultrastructural char- forming synaptic contacts with the CGCs.9 Evidence has acteristics also indicate that the CGCs may have mechanosen- shown that these fibers derive from the superior cervical gan- sory properties. The complex synaptic information points to a glion. The CGCs in the duck also receive calcitonin gene- specific regulative CGC function in parallel with ciliary muscle related peptide (CGRP)–positive efferent collaterals of trigem- contraction (accommodation). Axons originating from CGCs inal afferents that may indicate precentral reflex arcs.10 mainly supply the choroidal vasculature, thus implicating the Innervation of CGCs by postsynaptic sympathetic fibers in the CGCs as vasodilative neurons, but single CGCs may also inner- avian eye, however, indicates a more complex integrative func- vate other structures such as nonvascular choroidal smooth tion of the ganglion cell plexus, similar to that in the enteric muscle cells. (Invest Ophthalmol Vis Sci. 2004;45:361–367) nervous system.9 DOI:10.1167/iovs.03-0624 It is not yet known whether CGCs in primate eyes have a similar complex function. In the present study we investigated t is well established that the uvea in most species is inner- human CGCs to clarify which neurotransmitters are expressed Ivated by two parasympathetic pathways, namely by nerves by these cells, to characterize their presynaptic input, and to deriving from the ciliary ganglion and by those deriving from define their target tissue. the sphenopalatine ganglion. Within the uvea of human eyes, an additional group of nitric MATERIAL AND METHODS oxide synthase (NOS) and vasoactive intestinal peptide (VIP)- immunoreactive (IR) neurons has recently been discovered.1–3 The studies were performed in 50 human eyes with no previous clinical history of ocular disease, the donors ranging in age between 12 and 95 years. The eyes were obtained during autopsy 4 to 36 hours 1 2 after death. All donors had given permission to use their tissues for From the Departments of Anatomy II, and Anatomy I, Friedrich- research. Tissue observation was in accordance with the Declaration of Alexander University, Erlangen, Germany. Helsinki and the local regulations. Eyes with obvious signs of ocular Supported by German Research Community SFB 539 BII,2. Submitted for publication June 20, 2003; revised September 29, disease (e.g., macular degeneration, loss of retinal pigmented epithe- 2003; accepted October 26, 2003. lium) or ocular treatment other than artificial lens implantation (e.g., Disclosure: C.A. May, None; W. Neuhuber, None; E. Lu¨tjen- laser-treatment of the retina) were excluded from the study. A further Drecoll, None histologic screening for ocular disease was not performed. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertise- Immunohistochemistry ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. Corresponding author: Christian Albrecht May, Anatomisches Thirty-seven eyes of human donors ranging in age between 12 and 95 Institut II, Universita¨tsstr. 19, D-91054 Erlangen, Germany; years were observed 11 to 36 hours after death. All eyes were incised [email protected]. equatorially and fixed either for 4 hours in neutral buffered 4% para- Investigative Ophthalmology & Visual Science, February 2004, Vol. 45, No. 2 Copyright © Association for Research in Vision and Ophthalmology 361 Downloaded from iovs.arvojournals.org on 09/28/2021 362 May et al. IOVS, February 2004, Vol. 45, No. 2 TABLE 1. Specification of the Primary Antibodies Used in the Study Antibody Host Dilution Source Panneuronal marker PGP 9.5 Rabbit 1:200 Biotrend, Colone, Germany Tyrosine hydroxylase Rabbit 1:400 Chemicon, Hofheim, Germany Tyrosine hydroxylase Mouse 1:100 Chemicon Dopamine-␤-hydroxylase Rabbit 1:150 Affinity, Nottingham, UK Vesicular monoaminergic transporter 2 Rabbit 1:500 Biomar, Marburg, Germany Vesicular acetylcholine transporter Rabbit 1:1000 Biomar Neuronal nitric oxide synthase Rabbit 1:400 B. Mayer, Graz, Austria Vasoactive intestinal peptide Rabbit 1:400 Biotrend Neuropeptide Y Rabbit 1:200 Biotrend Substance P Rabbit 1:500 Biotrend Calcitonin gene-related peptide Rabbit 1:250 Affinity Galanin Rabbit 1:400 Chemicon Synaptophysin Mouse 1:500 Dako, Glostrup, Denmark Calretinin Rabbit 1:500 Biotrend Calretinin Goat 1:1000 Swant, Bellinzona, CH ␣-Smooth muscle actin Mouse 1:200 Sigma, St. Louis, USA Smooth muscle myosin Rabbit 1:250 Biotrend formaldehyde (PFA) or for 12 hours in Zamboni fixative, containing 4% thick) were performed and stained with toluidine blue. From localized PFA and 0.01% picric acid. The tissue was then rinsed in phosphate- CGCs, ultrathin sections were performed, stained with leaded citrate buffered saline (PBS, pH 7.4) several times. In 27 eyes, the posterior and uranyl acetate, and viewed with a transmission electron micro- segments were divided into four quadrants and wholemounts of the scope (model EM 902; Carl Zeiss Meditec, Oberkochen, Germany). choroid and sclera were performed. From some of the wholemounts and from the remaining eyes, serial 14- to 16-␮m-thick sagittal and ESULTS tangential sections were cut through the choroid and sclera with a R cryostat (Leica, Bensheim, Germany) and mounted on poly-L-lysine– Choroidal Ganglion Cells coated glass slides. Incubation with the primary antibody was performed overnight at In human choroidal sections and wholemounts incubated with room temperature, with the antibody diluted in PBS containing 1% antibodies against neuronal NOS (nNOS), all 1820 visualized bovine serum albumin and 0.1% Triton X. Control experiments were CGCs showed positive staining (Fig. 1A). No additional CGCs, performed by incubating the sections only with the dilution solution. either identified by their content of autofluorescent lipofuscin The primary antibodies used are listed in Table 1. The sections and or by a nonspecific light fluorescent background in the neuro- wholemounts were then rinsed in PBS and incubated for 1 hour with nal cytoplasm, were detected in the nNOS-stained specimens. an appropriate fluorescent-dye–conjugated secondary antibody, di- The nNOS staining was independent of the size (10–37 ␮min luted in PBS (Dianova, Hamburg, Germany). In the double-staining procedure, the steps were the same, but the incubation time of the primary antibody was reduced to 4 to 6 hours. Most of the antibodies showed sufficient staining in all specimens obtained, regardless of the donor age, time since death, or fixation. For some antibodies, certain special conditions must exist in the tissue to promote sufficient staining. Therefore, the number of tissue specimens was limited for these markers. The antibody against